1. Leptin Controls Parasympathetic Wiring of the Pancreas during Embryonic Life 
Sophie Croizier, Vincent Prevot, Sebastien G. Bouret 
Cell Reports 
Volume 15, Issue 1, Pages (April 2016)
DOI: /j.celrep
Copyright © 2016 The Authors Terms and Conditions

2. Figure 1
Ontogeny of the Parasympathetic Innervation of Pancreatic Islets
(A) Confocal images of VAChT-immunopositive fibers (red fluorescence) in pancreatic islets (insulin, green fluorescence) of E12, E15, and E18 WT embryos. Arrowheads point to VAChT fibers in close proximity to insulin-positive cells.
(B and C) Representative images showing VAChT immunoreactivity (red fluorescence) and its colocalization with synaptophysin1 labeling (turquoise fluorescence) in pancreatic β cells (insulin, green fluorescence) of (B) E18 WT mouse embryos and (C) 16-week-old ob/ob mice. Arrowheads point to double-labeled inputs (white labeling).
(D) Representative images of genetically labeled cell bodies (red fluorescence) in the hindbrain of postnatal day 0 (P0) Phox2b-Cre-tdTomato mice.
(E) Detail of Phox2b-Cre-tdTomato innervation of pancreatic islets (insulin, green fluorescence). Arrowheads point to td-Tomato-positive fibers that are also VAChT-positive. Arrows point to intrapancreatic ganglia containing tdTomato.
DMV, dorsomedial nucleus of the vagus nerve; NA, nucleus ambiguus; NTS, nucleus of the solitary tract; X, vagus nerve; 12N, hypoglossal nucleus; V4, fourth ventricle. Scale bars represent 100 μm (A), 20 μm (B and C), 200 μm (D), and 50 μm (E).
Cell Reports  , 36-44DOI: ( /j.celrep )
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3. Figure 2
Prenatal Leptin Exposure Impairs Parasympathetic Innervation of Pancreatic Islets
(A) Confocal images of VAChT-positive fibers (red fluorescence) in close proximity to insulin-positive cells (green fluorescence) in E12, E15, and E18 ob/ob embryos.
(B) Confocal images and quantification of the density of VAChT fibers (red fluorescence) innervating pancreatic β cells (insulin, green fluorescence) in E18 WT and ob/ob embryos that were injected with vehicle or leptin at E12 (1 μg per embryo) (n = 4 per group).
(C) Percentage change in the innervation of pancreatic islets by VAChT fibers between adult WT and ob/ob mice.
(D and E) Confocal images and quantification of pancreatic islet size (insulin, green fluorescence) and of the density of VAChT fibers (red fluorescence) innervating islets in 16-week-old (D) WT and (E) ob/ob mice that were injected with leptin or vehicle at E12 (n = 3–6 per group).
(F) Confocal images and quantification of the density of VIP-positive fibers (red fluorescence) innervating pancreatic β cells (insulin, green fluorescence) in 16-week-old WT and ob/ob mice that were injected with vehicle or leptin at E12 (1 μg per embryo) (n = 4 per group).
(G) Percentage change in the innervation of pancreatic islets by VIP fibers between adult WT and ob/ob mice.
(H) Photomicrographs and quantification of the β cell area in pancreases of 16-week-old ob/ob mice that were prenatally injected with leptin or vehicle (n = 4 per group).
Scale bars represent 100 μm (A–F) and 1 mm (G). ∗p < 0.05 versus WT (B) and versus WT + NaCl at E12 (D); ∗∗∗∗p < versus ob/ob (B), versus WT + NaCl at E12 (C), and versus ob/ob + NaCl at E12 (E and F); ####p < between the indicated groups (B). Values are shown as the mean ± SEM.
Cell Reports  , 36-44DOI: ( /j.celrep )
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4. Figure 3
Prenatal Leptin Exposure Causes Lifelong Dysregulation of Glucose Homeostasis
(A–D) Pre- (A and C) and post-weaning (B and D) growth curves (body weights) of WT (A and B) and ob/ob mice (C and D) that were injected with leptin or vehicle at E12 (n ≥ 10 per group).
(E–L) Glucose (E and I) and insulin (F and J) tolerance tests of 10- to 12-week-old ob/ob (E and F) and WT mice (I and J) that were injected with leptin or vehicle at E12 (n = 5–8 per group for the glucose tolerance test (GTT) and n = 7–8 per group for the insulin tolerance test [ITT]). Blood glucose levels in 13-week-old ob/ob (G) and WT mice (K) that were prenatally injected with leptin or vehicle (n = 3–7 per group). Plasma insulin levels in 16-week-old ob/ob (H) and WT (L) mice that were injected with leptin or vehicle at E12 (n = 5–7 per group).
(M and N) Glucose- (M) and L-arginine-stimulated (N) insulin secretion in adult ob/ob mice that were injected with leptin or vehicle at E12 (n = 3–5 per group).
(O) Pyruvate tolerance test of 8-week-old ob/ob mice that were prenatally injected with leptin or vehicle (n = 6 per group).
(P and Q) Plasma triglyceride (P) and free fatty acids (FFA) (Q) levels in 16-week-old ob/ob mice that were injected with leptin or vehicle at E12 (n = 4–7 per group).
∗p < 0.05 versus ob/ob + NaCl at E12 (M); ∗∗p < 0.01 versus ob/ob + NaCl at E12 (E); ∗∗∗p < versus ob/ob + NaCl at E12 (E) and versus WT + NaCl at E12 (J); and ∗∗∗∗p < versus WT + NaCl at E12 (J). Values are shown as the mean ± SEM.
Cell Reports  , 36-44DOI: ( /j.celrep )
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5. Figure 4
Leptin Blocks Cholinergic Axonal Growth from the Embryonic Hindbrain
(A) Representative photomicrographs of the hindbrain showing cell bodies (arrowheads) and vessels (arrows) labeled with fluorescent leptin (1ug per embryo, white labeling) 5 and 45 min after it was intracerebroventricularly injected into E12 embryos.
(B) Relative expression of Leprb mRNA in various brain areas and peripheral organs of E12 WT mouse embryos (n = 3 per group).
(C) Relative expression of Leprb mRNA in the hindbrain of E10, E12, and E14 WT mouse embryos (n = 3–5 per age). Numbers in bar graphs indicate Ct values.
(D) Confocal images of VAChT-immunoreactive cells (green fluorescence) and leptin-induced pSTAT3-immunopositive cells (red fluorescence) in the DMV of E12 WT embryos.
(E) Confocal images and quantification of the overall density of VAChT-positive fibers in organotypic cultures of isolated E12 hindbrains. The cultures were incubated with leptin (100 ng/ml) or vehicle for 48 hr (n = 5–6 explants per group).
DMV, dorsomedial nucleus of the vagus nerve; HIND, hindbrain; V4, fourth ventricle. Scale bar, 100 μm. ∗∗∗p < versus vehicle (E); and ∗∗∗∗p < between indicated groups (D). Values are shown as the mean ± SEM.
Cell Reports  , 36-44DOI: ( /j.celrep )
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6. Cell Reports 2016 15, 36-44DOI: (10.1016/j.celrep.2016.02.088)
Copyright © 2016 The Authors Terms and Conditions